Physiological,Biochemical And Gene Expression Response Of Sugarcane To Sporisorium Scitamineum Infection

Sugarcane smut,which is caused by Sporisorium scitamineum,poses a serious threat to the sugarcane industry by significantly decreasing yield and sucrose content,and is one of the most severe fungal diseases in sugarcane planting areas worldwide.Due to the time-consuming investigation cycles of long latency time after the invasion of smut pathogen and at least two crop seasons in field evaluation of the smut disease,sugarcane smut resistance assessment can only be conducted at few sugarcane clones.It lead to low efficiency and has only a little chance for selection smut resistant lines.Hence,it is important to establish an efficient but time-saving smut resistance assessment technique.Based on the previous studies,a total of nine sugarcane varieties with different phenotypic resistance levels were used for investigation.After challenge with smut pathogen by artificial needle puncture inoculation of sugarcane buds and cultured in a controllable environment,sugarcane buds were collected at different time points(0 d,1 d,3 d and 7 d)for analysis of copy number change of smut pathogen and sugarcane physiological,biochemical and gene expression response to S.scitamineum infection,and evaluation of its smut resistance.The main target in this study is to establish a new sugarcane smut resistant evaluation system.Furthermore,peroxidase genes in sugarcane responsive to smut pathogen challenge were identified,which will provide basic information for development of functionl markers for smut resistance breeding.The main results and conclusions were as follows.1.Nine sugarcane varieties with different phenotypic resistance levels were selected,and TaqMan quantitative real-time polymerase chain reaction(qRT-PCR)analysis was performed to measure copy number changes of smut pathogen in sugarcane buds at four different time points(0-7 d)after needle puncture inoculation.The results indicated that the copy number of smut pathogen in sugarcane bud was increased with the extension of inoculation time and reached a peak value(between 2.49× 10~5-6.09× 10~6 copies/μL)on 7 d.There were significant differences between different time points in all tested genotypes,suggesting it was feasibility via this assessment technique for detecting the copy number of smut pathogen during early inoculation stages,which may serve to access the smut resistance.The amount of S.scitamineum among the nine genotypes decreased in the following order:YZ03-258<FN40<YZ01-1413<GT02-467<ROC22<YT96-86<YZ03-103<FN39<LC05-136.Except for three genotypes of LC05-136,YT96-86 and FN40,the copy numbers of smut pathogens were generally in the same order as the incidences of field smut disease in the other six sugarcane genotypes(YZ03-258<YZ01-1413<LC05-136<YT96-86<GT02-467<ROC22<FN39<YZ03-103<FN40).That is,lower resistance with higher copy number.Moreover,the activity of ascorbate peroxidase(APX),catalase(CAT),peroxidase(POD),superoxide dismutase(SOD),β-1,3-glucanase and the content of malondialdehyde(MDA)in the tested varieties at 4 time points were measured.The results based on correlation test showed there were significant positive correlation(p≤0.05)between POD and β-1,3-glucanase,and APX and β-1,3-glucanase.Principal component analysis indicated that POD,APX,CAT,SOD,β-1,3-glucanase,and MDA were grouped into three main components:the first principal components including POD,APX,CAT and SOD,the second and the third only contain β-1,3-glucanase and MDA,respectively,and the cumulative contribution rate(80.177%)was more than 80.0%,indicating that these parameters could be used as physiological and biochemical indicators of smut resistance in sugarcane.Based on the physiological and biochemical parameters obtained above,subordinate function method was furtherly used to assess the smut resistance and the order of these nine genotypes was as follows:YZ03-258>FN40>YZ01-1413>GT02-467>ROC22>YZ03-103>YT96-86>FN39>LC05-136,which was in agreement with the results of copy number determination of smut pathogens.The results suggest that an accessment of sugarcane smut resistance in time-serving way is possible by a comprehensive analysis of the copy numbers of smut pathogen and the values of 6 parameters of POD,APX,CAT,SOD,β-1,3-glucanase and MDA after artificial needle inoculation and cultured in a controllable environment.2.APX is an important enzyme during the process of active oxygen scavenging.In this study,the APX activity in both sugarcane varieties Yacheng05-179(smut resistant)and Liucheng03-182(smut susceptible)challenged with S.scitamineum for 2 d was measured,which showed a significantly higher in the resistant variety than that in the susceptible one.An APX gene with length of 1 171 bp and encoding 345 amino acids,named ScAPX(GenBank No.KJ7565501),was separated from sugarcane by conducting electronic cloning and RT-PCR.ScAPX contained no signal peptide which was speculated that it was non-secretory protein and was most likely located in the matrix of mitochondrial(91.1%)or chloroplast(88.7%).The APX protein sequence homology among ScAPX and sorghum(GenBank Accession No.XP002447862.1)was up to 97%.ScAPX is a constitutive expression gene and can express in root,bud,leaf,stem skin and stem pith.The results of tissue specificity analysis showed that the highest expression level of ScAPX was in stem skin which was 19.7 times of that in leaf.The expression peak under salicylic acid(SA),methyl jasmonate(MeJA)and hydrogen peroxide(H2O2)(group 1)stresses appeared earlier than those under abscisic acid(ABA),sodium chloride(NaCl)and polyethylene glycol 8000(PEG 8000).Futhermore,ScAPX expression in group l appeared to increase beginning at preliminary stage and gradually decrease after reaching the peak,while the situation was different for the ScAPX expression maintaining stable after reaching the peak under ABA,NaCl and PEG 8000 stresses.Though the gene expression under the exogenous stresses are different,ScAPX is positive response to the stresses of SA,MeJA,H2O2,ABA,NaCl and PEG 8000.3.Plant class III peroxidases(PODs,E.C 1.11.1.7),which are specific in plant species,organs and developmental stage,play diverse roles in the growth and development of plants.In this study,two full length cDNA of ScPOD01 and ScPOD02(GenBank No.KU298453 and KU593507)and DNA(GenBank No.KU311043 and KU593508)sequences were isolated from smut resistant genotype Yacheng05-179 in sugarcane buds challenged with S,scitamineum for 2 d.According to the sequencing and alignment,ScPODO 1 contains four extrons and three introns typical POD structure,while ScPOD02 just contains one intron.The phylogenetic tree of PODs demonstrated clearly that sugarcane ScPOD01 encoded a putative class III POD and ScPOD01 belonged to class I.The gene expression patterns of ScPOD01 and ScPOD02 under various abiotic stresses were revealed by RT-qPCR,and their distinctive difference response patterns were observed.Besides SA,ScPOD01 was positive response to MeJA,ABA,H202,NaCl and PEG 8000 stresses,while different for ScPOD01,indicating the different role in function.The proteins of ScPOD01 and ScPOD02 expressed in prokaryotic cells were about 55 kDa and 60 kDa,respectively.The vector containing ScPOD01 named as pCAMBIA 1300-ScPOD01,was constructed and introducted into Agrobacterium tumefaciens and transient expression in Nicotiana benthamiana was conducted by injecting the cells of A.tumefaciens.A deeper DAB staining color in N.benthamiana leaves was observed.Meanwhile,gene expression of ScPOD01 and four immunity related marker genes in the N.benthamiana leaves,including HR related gene NtHSR201和 NtHSR203 and a JA related gene NtPR-la/c(positively response)were up regulated,an ET synthesis depended genes NtEFE26 was down regulated,which can be further proved that ScPOD01 was a SA-insensitive POD.The deeper DAB staining color was also found in N.benthamiana leaves after the transient over-expression of ScPOD01 in N.benthamiana.Meanwhile,the ScPOD01 expression and HR marker genes(NtHSR201 and NtHSR203),ET synthesis dependent genes(NtEFE26 and NtAccdeaminase)were all up-regulated.Subcellular localization analysis indicated that ScPOD01 was located in the plasma membrane or cell wall,suggesting that ScPOD01 belongs to a wall-type PODs,which may be related to cell defense function.4.RT-qPCR was performed to analysis the gene expression levels of a total of 7 sugarcane genes,including two β-1,3-glucanase genes of ScGluAl and ScG48(GenBank No.KC848050 and KF664181),two CAT genes of ScCATl and ScCAT2(GenBank No.KF664183 and KF528830)isolated in our previous studies,and ScAPX,ScPOD01 and ScPOD01 isolated in this work,in nine sugarcane genotypes after smut pathogen infection.The gene expression patterns of ScCAT1,ScCAT2,ScPOD01,ScPOD02 and ScAPX in 9 sugarcane genotypes were different and the response trends to smut pathogen stress were not obvious.While,ScGluAl gene was significantly response to S.scitamineum stimulis,as its gene expression level was higher in YZ03-258,LC05-136(smut resistant)and FN39(smut middle susceptible)varieties than smut susceptible ones,and the gene expressions amost appeared to be increased in various sugarcane genotypes and higher than that in the control at different time points.Meanwhile,high expression file of ScG48 was earlier detected in response to smut fungus in the sugarcane resistant varieties,and the response time was delayed in the medium-and susceptible-cultivars.Results suggesting that ScGluA1and ScG48 can be expected as candidate genes for the development of functional marker genes in sugarcane smut resistant breeding.Its further evaluation was needed.